Conserved Noncoding Sequences Regulate <i>lhx5</i> Expression in the Zebrafish Forebrain

<div><p>The LIM homeobox family protein Lhx5 plays important roles in forebrain development in the vertebrates. The <i>lhx5</i> gene exhibits complex temporal and spatial expression patterns during early development but its transcriptional regulation mechanisms are not well understood. Here, we have used transgenesis in zebrafish in order to define regulatory elements that drive <i>lhx5</i> expression in the forebrain. Through comparative genomic analysis we identified 10 non-coding sequences conserved in five teleost species. We next examined the enhancer activities of these conserved non-coding sequences with Tol2 transposon mediated transgenesis. We found a proximately located enhancer gave rise to robust reporter EGFP expression in the forebrain regions. In addition, we identified an enhancer located at approximately 50 kb upstream of <i>lhx5</i> coding region that is responsible for reporter gene expression in the hypothalamus. We also identify an enhancer located approximately 40 kb upstream of the <i>lhx5</i> coding region that is required for expression in the prethalamus (ventral thalamus). Together our results suggest discrete enhancer elements control <i>lhx5</i> expression in different regions of the forebrain.</p></div

CNS2 contains hypothalamic enhancer activity and responses to FGF signaling.

<p>(<b>A-B</b>) Double in situ hybridization results indicate CNS2 contains hypothalamic enhancer activity. The hypothalamic marker <i>nkx2</i>.<i>1a</i> and <i>nkx2</i>.<i>2b</i> are stained in dark blue, reporter <i>egfp</i> stained in red. (<b>C-D</b>) SU5402 treatment severely reduces CNS2 activity. Vehicle DMSO treated embryos show restricted hypothalamic EGFP reporter expression (pointed by the arrow in C). Embryos treated with the FGF signaling inhibitor SU5402 during the segmentation stage (10-24hpf) show minimal EGFP signals in the hypothalamic region (arrow in D, n = 48/55). (<b>E-F</b>) SU5402 treatment down-regulates endogenous <i>lhx5</i> expression in the hypothalamic region. Endogenous <i>lhx5</i> shows robust expression in the hypothalamic region (pointed by the arrow in E). SU5402 treatment during the segmentation stage down-regulates <i>lhx5</i> expression in the hypothalamic region (arrow in F, n = 25/28). (<b>G</b>) Multiple sequence alignments of the CNS2 region in the five teleost species. The identified FGF downstream factor Pea3 binding site is highlighted in blue. Lateral view of the forebrain regions of embryos at 24 hpf (A-F), anterior to the left. Scale bar: 40μm in A-B; 50μm in C-D.</p

Region specific enhancer activity of the identified CNSs.

<p>(<b>A-B</b>) CNS8 and CNS9, located in the vicinity of the <i>lhx5</i> promoter region give rise to broad reporter EGFP expression in the forebrain regions. (<b>C</b>) CNS2 located approximately 50 kb upstream of the <i>lhx5</i> coding region gives rise to restricted EGFP signal in the anterior ventral forebrain. (<b>D</b>) CNS4 located 40 kb upstream of the <i>lhx5</i> coding region, gives rise to restricted EGFP expression in the diencephalic region. (<b>E</b>) Vector construct gives rise to basal non-tissue specific EGFP expression in transient expression assay. Lateral view of the forebrain regions of embryos at 24 hpf, anterior to the left. Scale bar: 50μm.</p

In-Vehicle Exposures to Particulate Air Pollution in Canadian Metropolitan Areas: The Urban Transportation Exposure Study

Commuters may be exposed to increased levels of traffic-related air pollution owing to close proximity to traffic-emissions. We collected in-vehicle and roof-top air pollution measurements over 238 commutes in Montreal, Toronto, and Vancouver, Canada between 2010 and 2013. Voice recordings were used to collect real-time information on traffic density and the presence of diesel vehicles and multivariable linear regression models were used to estimate the impact of these factors on in-vehicle pollutant concentrations (and indoor/outdoor ratios) along with parameters for road type, land use, and meteorology. In-vehicle PM_2.5 and NO₂ concentrations consistently exceeded regional outdoor levels and each unit increase in the rate of encountering diesel vehicles (count/min) was associated with substantial increases (>100%) in in-vehicle concentrations of ultrafine particles (UFPs), black carbon, and PM_2.5 as well as strong increases (>15%) in indoor/outdoor ratios. A model based on meteorology and the length of highway roads within a 500 m buffer explained 53% of the variation in in-vehicle UFPs; however, models for PM_2.5 (<i>R</i>² = 0.24) and black carbon (<i>R</i>² = 0.30) did not perform as well. Our findings suggest that vehicle commuters experience increased exposure to air pollutants and that traffic characteristics, land use, road types, and meteorology are important determinants of these exposures

Additional file 1: of Facilitators and barriers to using physical activity smartphone apps among Chinese patients with chronic diseases

Survey of Physical Activity App Use Among Chronic Disease Patients (English Version). The questionnaire describes the participants’ demographic profile, patient health status and current status of doing exercise, current status of using activity apps, and willingness of and barriers to using physical activity apps. (PDF 138 kb

Inhibition of Dcc function causes ADt axons to project dorsally or to form multiple processes.

<p>(<b>A</b>) Labeling of individual ADt neurons by mosaic expression of fluorescent protein tdTomato. Image of a live 36 hpf <i>Tg(lhx5BAC:Kaede)</i> animal that was injected with <i>emx3:Gal4FF</i> and <i>UAS:tdTomato</i> plasmids is shown. The tdTomato labeled neuron projected an axon into the AC. Merge panel shows the position of the tdTomato labeled soma (marked by an arrowhead). Scale bar = 50 µm. (<b>B</b>) Injection of <i>dcc</i> morpholino causes ADt neurons to project axons dorsally or to form multiple processes. Labeled ADt neurons are marked by arrowheads in the merged panels. Left panels show an ADt neuron with a normal ventrally projecting axon in a control animal. Middle panels show an ADt neuron with an aberrant dorsally projecting axon in a <i>dcc</i>-MO injected animal. Right panels show an ADt neuron with both ventrally and dorsally projecting processes. Black arrow in the single slice images indicates the origin of the axon on the surface of the cell body. Red bar indicates the middle of the dorsal and the ventral side of the labeled neuron cell body. Scale bar equals to 20 µm in the projected images or 10 µm in the single slice images. (<b>C</b>) Additional examples of ADt neurons with multiple aberrant axons in <i>dcc</i>-MO injected animals. Scale bar = 15 µm.</p

Expression patterns of <i>dcc</i> and <i>netrin</i>.

<p>(<b>A</b>) <i>dcc</i> is expressed in the dorsal telencephalic region at 24 hpf and 36 hpf. (<b>B</b>) <i>dcc</i> and <i>lhx5</i> are co-expressed in the dorsal telencephalon at 20 hpf. ADt neurons are migrating from their medial positions in the neural tube to lateral positions at 20 hpf. Scale bar: 100 µm for lateral view; 60 µm for frontal view.</p

Dcc is required for correct asymmetric outgrowth of ADt neuronal axons.

<p>(<b>A</b>) Dcc expression was reduced after injection of <i>dcc</i> translation-blocking morpholinos into zebrafish embryos. Endogenous Dcc protein was detected as a band of approximately 170 kb. Tubulin served as a loading control. M: size marker. (<b>B</b>) ADt neurons project axons dorsally when Dcc function is inhibited by morpholino injection. Images of live animals were acquired as in Fig. 1C. The pixel intensity value of aberrant axon is shown in the bottom left corner of each panel. Scale bar = 50 µm. (<b>C</b>) Quantitation of ADt neuronal axon defects. Horizontal axis shows the treatment group labels and vertical axis shows the percentage of embryos in each phenotypic category (Grade 0–3) for each treatment group. Numbers inside parentheses denote numbers of animals analyzed for each treatment group. Asterisks and brackets represent <i>p</i><0.05 by Mann-Whitney U test.</p

Effects of inhibition of Netrin1 or Neogenin1 function on the ADt axons.

<p>(<b>A</b>) ADt neurons project axons dorsally when Netrin1 function is inhibited (<i>ntn</i>-MO). Knockdown of Neogenin1 function doesn’t cause ADt neuron to project axon dorsally (<i>neo</i>-MO). Images were processed as in Fig. 3B. The pixel intensity value of aberrant axon is shown in the bottom left corner of each panel. Scale bar = 50 µm. (<b>B</b>) Quantitation of ADt neuronal axon defects. Horizontal axis shows the treatment group labels and vertical axis shows the percentage of embryos in each phenotypic category (Grade 0–3) for each treatment group. Numbers inside parentheses denote numbers of animals analyzed for each treatment group. (<b>C</b>) Synergistic effects between sub-threshold Dcc-Netrin1 and Dcc-Neogenin morpholino knockdowns. <i>dcc</i>-sub_MO and <i>ntn</i>-sub_MO: sub-threshold concentration morpholino. At least three independent injections were performed for each treatment group. Numbers inside parentheses denote numbers of animals analyzed. Asterisks represent <i>p</i><0.001 by ANOVA test.</p

Metro Commuter Exposures to Particulate Air Pollution and PM_2.5-Associated Elements in Three Canadian Cities: The Urban Transportation Exposure Study

System-representative commuter air pollution exposure data were collected for the metro systems of Toronto, Montreal, and Vancouver, Canada. Pollutants measured included PM_2.5 (PM = particulate matter), PM₁₀, ultrafine particles, black carbon, and the elemental composition of PM_2.5. Sampling over three weeks was conducted in summer and winter for each city and covered each system on a daily basis. Mixed-effect linear regression models were used to identify system features related to particulate exposures. Ambient levels of PM_2.5 and its elemental components were compared to those of the metro in each city. A microenvironmental exposure model was used to estimate the contribution of a 70 min metro commute to daily mean exposure to PM_2.5 elemental and mass concentrations. Time spent in the metro was estimated to contribute the majority of daily exposure to several metallic elements of PM_2.5 and 21.2%, 11.3% and 11.5% of daily PM_2.5 exposure in Toronto, Montreal, and Vancouver, respectively. Findings suggest that particle air pollutant levels in Canadian metros are substantially impacted by the systems themselves, are highly enriched in steel-based elements, and can contribute a large portion of PM_2.5 and its elemental components to a metro commuter’s daily exposure